The need to rapidly extract and process genomic material for identification of biological agents and disease is an ever increasing requirement. Methods that allow the complete unbiased isolation of nucleic acids from microorganisms and cellular samples are necessary to aid in molecular analysis methods and detection schemes. The biases implicit in samples makes it essential to develop preparation methods that directly access the nucleic acid content for field portable systems. This requires overcoming a variety of interferences that diminish quality, yield, and diversity of extracted nucleic acids. Routine laboratory methods for cell lysis include freeze/thaw, proteinase K, lysozyme, and guanidium salt treatments followed by ethanol or 2-propanol precipitation of liberated DNA; ballistic disintegration; and sonication at low frequencies (e.g., kilohertz) after pretreatment with other chemicals. See D. P. Chandler et al., Anal. Chem. 73, 3784 (2001).
For field portable systems, the tendency has been to simply downscale laboratory-scale equipment in genetic engineering to microscale on-chip processing. Therefore, a key aspect is that the extraction format must be highly scalable to benefit from many types of biodetection systems that are already in place. Many of the current nucleic acid extraction techniques, however, require significant manual intervention and consumables leading to limitations that are especially relevant for the unattended, timely detection of biological warfare agents or other microorganisms in a complex milieu. The continued reliance on large laboratory equipment (e.g., centrifuges, gel electrophoresis units, ultracentrifuges), requirements for chemical or enzymes that are labile or need special handling, and storage and disposal, further impede progress toward miniaturized autonomous detection.
Microsystems have been developed that use mechanical, chemical, thermal, and chemical methods for cell lysis. Ultrasonic waves are known to induce significant pressure variation and induce cavitation within fluids. See G. Zhang et al., Jpn. J. Appl. Phys. 35, 3248 (1996). Thus, acoustic waves can provide a non-invasive lysing mechanism which is compatible with sealed microsystems. Acoustic methods avoid the use of harsh chemicals which often interfere with subsequent detection methods (e.g., PCR). Often the altered pH and chemical background adds additional steps that can otherwise be avoided. Recently, large-scale acoustic transducers have proven powerful for disrupting cell membranes and spores and releasing the contents of the cytoplasm for subsequent DNA analysis. See D. P. Chandler et al.; and P. Belgrader et al., Anal. Chem. 71, 4232 (1999). Thin-film based ultrasonic actuators have also been used to lyse cellular samples and have proven effective for microsystem applications. See T. C. Marentis et al., Ultrasound in Med. & Biol. 31, 1265 (2005); and H. Jagannathan et al., IEEE Ultrason. Symp., 859 (2001). However, small-scale actuators suffer from limitations in attainable film thickness and meeting the thermal requirements for long-term use. The deposition thicknesses that can be reasonably attained are between 1 to 10 which translates to range of 316 MHz to 3.165 GHz for ZnO. However, a frequency of less than 300 MHz is far more optimal for coupling into the fluid. Moreover, these devices must be strictly operated in a pulsed mode to prevent device damage.
A second major problem is that nearly all microsonicator approaches lack on-chip nucleic acid extraction processing capability. Thus, the lysate must be processed and purified off-chip, reducing the effectiveness of a microsystem solution. Nucleic acid purification methods that are suitable for on-chip applications require the use of silicon based microstructures, commercial nucleic acid binding media, silica beads in the presence of chaotropic salts, and silica matrices. See N. C. Cady et al., Biosens. Bioelectr. 19, 59 (2003); M. Moré et al., Appl. Environ. Microbiol. 60, 1572 (1994); R. Boom et al., J. Clin. Microbiol. 28, 495 (1990); and K. Wolfe et al., Electrophoresis 23, 727 (2002). Though packed silica beads bind and elute nucleic acids, their inherent instability due to compression causes widely varying results. This limitation has been overcome by using a gelled sol-gel solution of silica beads to stabilize the matrix, improving reproducibility. Recently another powerful nucleic acid extraction method has been demonstrated which uses NAFION coated gold films to reversibly capture nucleic acids. See M. Lee et al., Anal. Biochem. 380, 335 (2008). This method is particularly easy to implement in microfluidic format and only needs low DC voltages for operation.
Therefore, a need remains for an acoustic-based microfluidic lysing device that can be integrated with an on-chip nucleic acid extraction processing capability and can be used in a field portable system.